1. Field of the Invention
The present invention relates to compositions and uses of modulators of annexin and of cartilage homeostasis, including therapeutic uses in the treatment and prevention of arthritis and arthritic disease.
2. Background Art
One in seven Americans have some form of degenerative joint disease, including arthritis or arthritic disease. Arthritis and arthritic diseases include over one hundred different types of joint disease, including, for example, non-inflammatory forms, like osteoarthritis (OA), and inflammatory forms, like rheumatoid arthritis (RA). OA is the most common form of arthritis or arthritic disease, affecting over 16 million people in the United States. OA is thought to be a consequence of an attempted but failed repair of damaged cartilage tissue [3]. RA, considered to be an autoimmune disease having an unknown etiology, affects 2.1 million Americans.
In various types of arthritis and arthritic diseases, the steady-state metabolism for collagen in the extracellular matrix of articular cartilage is disturbed. This disturbance causes more degradation than synthesis of collagen, resulting in a net loss in cartilage at joint surfaces and an interruption of cartilage homeostasis. Thus, arthritis and arthritic diseases, like OA and RA, are characterized by localized proteoglycan and cartilage loss and an accompanying bone response. It has been hypothesized that destruction of collagenous extracellular matrix in subjects with arthritis is augmented by the accumulation of specific catabolic fragments of cartilage collagens that shut down matrix synthesis and activate matrix metalloproteinases [5]. There is no known cure for arthritis, and treatment is generally aimed at symptomatic relief of joint pain and stiffness using analgesics, anti-inflammatories, or injectable corticosteriods.
Annexins constitute a large family of calcium and phospholipid binding proteins. At least one example of an annexin is expressed in almost every mammalian, insect, plant, and primitive eukaryotic cell type, but no single function has been identified [1]. Calcium binding sites appear to be conserved across the various annexins.
Benzodiazepines are known to bind with high affinity to GABAA receptors in the brain and to influence chloride channel opening. The effects of various benzodiazepine derivatives include, for example, anxiolytic activities, anticonvulsant effects, cholecystokinin receptor antagonist, opiate receptor ligand, platelet-activating factor ligand, human immunodeficiency virus trans-activator Tat antagonist, and reverse transcriptase inhibitors. Recently, the in vitro interactions between various annexins and various benzodiazepine derivatives have been studied [2]. Different effects in liposomes were observed with the various benzodiazepine derivatives and the various annexins. For example, BDA452 (3-(R,S)-(L-tryptophanyl)-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one)inhibited calcium fluxes induced by annexins V, V-N1, VIa, and VIb but increased calcium fluxes induced by annexins I, III, and VI; whereas, BDA 250 (1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one)) had no effect on annexin V-induced calcium influx but inhibited influx induced by annexins I and III [2]. No such benzodiazepine effect has been shown in intact cells, however, and there has been no suggestion that benzodiazepines may influence cartilage homeostasis or repair.
The present invention provides a method of treating a subject with arthritis or an arthritic disease or preventing arthritis or arthritic disease in a subject, comprising administering to the subject a therapeutically effective amount of an agent that attenuates annexin function.
Also provided are methods of screening for agents that increase collagen synthesis, decrease collagen degradation, or reduce or delay apoptosis, and methods of using the identified agents for attaining or maintaining cartilage homeostasis, promoting cartilage repair, increasing collagen synthesis, decreasing collagen degradation, or reducing or delaying apoptosis.
The invention also provides a method of decreasing mineralization of vesicles derived from chondrocytes, comprising contacting the vesicles with an agent that attenuates annexin function, and a method of promoting endochondral bone growth in a subject, comprising administering to the subject a therapeutically effective amount of an agent that attenuates annexin function.
The present invention also provides methods of screening for genes involved in modulating collagen synthesis using differential gene expression methods.
Also provided by the present invention is a method of treating a subject with arthritis or arthritic disease, comprising administering to the subject a nucleic acid that either blocks expression of the gene identified by the screening method and further identified as decreasing collagen synthesis or increasing collagen degradation, whereby cartilage homeostasis is achieved, cartilage repair is promoted, collagen synthesis is increased, or collagen degradation is decreased; or promotes expression of the gene identified by the screening method of the invention and further identified as increasing collagen synthesis or decreasing collagen degradation, whereby the nucleic acid is expressed in a cell and whereby cartilage homeostasis is achieved, collagen synthesis is increased, or collagen degradation is decreased.
The present invention also provides a composition comprising isolated chondrocytes, an agent that attenuates annexin function, and collagen fragments or other means for challenging chondrocytes, including contact with an agent that inhibits cell attachment, downregulates collagen synthesis, activates metalloproteinases, or induces cartilage breakdown, including for example, collagen fragments, collagen peptides, or immune mediators.